Project Summary Influenza A virus causes disease in 5-20% of the population with over 200,000 hospitalizations annually in US. The antigen drift and shift of influenza virus pose a serious challenge for flu vaccination program. Although IFN response is known as the first important host defending mechanism to control influenza virus replication at different steps, influenza virus has acquired many strategies to suppress/antagonize IFN function effectively allowing its replication in the hosts. It is well known that influenza viral nonstructural protein 1 antagonizes IFN function through suppressing IFN production. However, less is known on how influenza virus counteracts function of IFN-stimulated genes (ISGs), many of which have antiviral functions. Our recently developed a quantitative and high-throughput genomics system has enabled the identification of novel IFN-sensitive mutations on multiple viral segments across influenza genome. The goal of this project is to determine how influenza virus antagonizes host anti-viral mechanism by studying recently identified novel IFN-sensitive mutations using a comprehensive genomic and proteomic platform in vitro and in vivo. The research plan focuses on three motifs on influenza viral proteins PA, PB1, and M1. Each motif, represented by multiple mutations located together in a small surface area, might target a cellular protein or step of IFNR signaling pathway. In addition, our preliminary data suggest mutations on the M1 motif interact with IFN production pathway, while motifs on PA and PB1 may counteract ISGs downstream of IFN production. The hypothesis is that these three anti-IFN motifs of influenza PA, PB1, and M1 interact with cellular proteins to antagonize IFN functions. A series of complementary approaches including high-density mutagenesis, mRNA display-deep sequencing, ISG cDNA library screening, CRISPR/Cas9 gene editing, human lung primary culture system, and gene knockout mice are proposed in three independent while logically connected Specific Aims (SAs) to test our scientific hypothesis. SA#1 is to define the motifs on viral proteins PA, PB1, and M1 that are critical for the anti-IFN functions. SA#2 is to utilize WT and mutant viral proteins containing IFN-sensitive mutations to determine the cellular proteins interacting with the anti-IFN motifs. In SA#3 is to determine the functional interaction and its biological impact in vitro and in vivo. The completion of proposed project will reveal mechanism for the anti-IFN strategy used by influenza virus, advance our understanding of influenza pathogenesis, and provide valuable targets for more effective influenza vaccine and anti-viral therapy.